Abstract
Chemotherapeutics are widely used in cancer treatments, but their toxicity, bioavailability and solubility present challenges. RNA nanotechnology has emerged as a promising modality for targeted delivery of chemotherapeutics. Structurally, RNA is thermostable, while conformationally it is dynamic and flexible. RNA’s unique deformability and motility lead to rapid spontaneous tumor accumulation and glomerular excretion, thus fast body clearance, while its anionic charge and favorable small size prevent accumulation in vital organs, resulting in undetectable toxicity. We developed branched 4-way junction (4WJ) nanoparticles that were stable with a melting temperature >80 °C, even when conjugated with 24 drugs per 4WJ. Each 4WJ RNA component strand can conjugate six molecules of hydrophobic chemotherapeutic drugs, such as camptothecin, paclitaxel and SN-38. Thus, each 4WJ carries a total of 24 drug molecules spaced to prevent aggregation. RNA conjugation improved paclitaxel water solubility 32,000-fold. This protocol describes the construction of 4WJ RNA drug complexes for cancer therapy. Specific procedures include the modification of chemical drugs, conjugation of multiple prodrug molecules to each synthesized RNA component strand, assembly of RNA nanoparticles and their purification and characterization. Prodrugs are conjugated to RNA nanoparticles via efficient click chemistry, creating an ester linker that is cleaved by esterases in tumor tissues or cells, allowing the prodrugs to return back to their original structures and chemistry upon delivery and release, minimizing toxicity. Inclusion of tumor targeting ligands demonstrated specific delivery of high payload chemotherapeutics to tumors, controlled release of chemical drugs and strong tumor inhibition.
Key points
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RNA strands containing modified nucleotides are produced by using solid-phase synthesis for conjugation with multiple chemotherapeutic prodrugs, including camptothecin, paclitaxel and SN38, by copper-chelated click chemistry, where copper is easily removed during purification due to its much smaller size compared to RNA before assembling the 4WJ RNA nanoparticles, which can be loaded with up to 24 drug molecules per nanoparticle.
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The RNA nanoparticles can be designed to contain a tumor-targeting ligand, resulting in specific uptake by the target cell type, while the drug payload is released via cleavage by esterases in the tumor microenvironment to ensure site-specific delivery.
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Acknowledgements
We thank J. Carroll for his comments on this manuscript ahead of submission. This Protocol is a comprehensive outcome supported by many years of funding from the NIH. During the preparation and revision of this manuscript, T.Y. and K.J. were supported in part by the President Research Excellence Catalyst Award to P.G. from OSU; and effort in part to P.G. and D.W.B. by R01CA293945 and R01CA257961, respectively.
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P.G. and D.W.B. conceived and led the project. D.W.B., K.J. and Y.T. designed and conducted the experiments and co-wrote the manuscript with P.G.
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P.G. is the licenser of Oxford Nanopore Technologies; co-founder of ExonanoRNA, LLC; and a consultant for RNA Nanobiotics.
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Nature Protocols thanks Yuting Wen, Alex G. Hamilton, Xiangang Huang, Jinhwan Kim, Jong Bum Lee and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references
Guo, S. et al. Nat. Commun. 11, 972–982 (2020): https://doi.org/10.1038/s41467-020-14780-5
Piao, X. et al. Adv. Sci. 6, 1900951 (2019): https://doi.org/10.1002/advs.201900951
Li, X. et al. Biomaterials 305, 122432 (2023): https://doi.org/10.1016/j.biomaterials.2023.122432
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Binzel, D.W., Jin, K., Yudhistira, T. et al. Conjugation of hydrophobic drugs to motile pRNA 4WJ nanoparticles for spontaneous tumor targeting and undetectable toxicity. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01306-w
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DOI: https://doi.org/10.1038/s41596-025-01306-w
